Apparatus for demineralizing osteoinductive bone

ABSTRACT

The invention is directed to an apparatus for producing demineralized osteoinductive bone. The apparatus demineralizes bone by subjecting bone, including, for example, ground bone, bone cubes, chips, strips, or essentially intact bone, to either a rapid high volume pulsatile acidification wave process or to a rapid continuous acid demineralization process. The pulsatile acidification wave process includes subjecting bone to two or more rapid pulse/drain cycles in which one or more demineralizing acids is rapidly pulsed into a vessel containing bone, and after a desired period of time, is rapidly drained from the vessel. The continuous acid demineralization process includes subjecting bone to a continuous exchange of demineralizing acid solution in which the demineralizing acid solution is recirculated from the container holding the bone through an ion exchange media. Calcium and phosphate are thereby removed from the bone to produce a regenerated acid, and the regenerated acid is returned to the container holding the bone. Both processes allow bone to be rapidly demineralized to a precise and specific desired residual calcium level without sacrificing osteoinductivity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.10/180,989, filed Jun. 26, 2002, which is a continuation-in-part ofapplication Ser. No. 09/655,711, filed Sep. 5, 2002, which issued asU.S. Pat. No. 6,534,095 on Mar. 18, 2003, and which claims the benefitof provisional application No. 60/152,272, filed Sep. 3, 1999, both ofwhich are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the preparation of bone grafts. Thepresent invention further relates to the demineralization ofosteoinductive bone.

BACKGROUND OF THE INVENTION

Demineralized freeze-dried bone allograft is widely used in the repairof skeletal defects and periodontal disease. It is known that theimplantation of acid demineralized bone in the form of a powder inextraskeletal sites may stimulate new bone formation. Various groupsincluding Syftestad, 1982; Urist et al., 1967; Urist and Strates, 1979;Urist and Strates, 1971; and Urist et al., 1983 have suggested that anoncollagenous protein or proteins present in demineralized bone has theability to induce new bone formation when present within the implantedbone matrix.

Current procedures used to demineralize ground bone involve the use ofethanol to remove lipids and hydrochloric acid to remove the mineralcomponents of bone. These known methods are problematic in that theyrequire prohibitively long periods of time for processing resulting in avery low demineralization rate, require excessive handling of the groundbone being processed, are capable of processing only small amounts ofground bone, and result in a demineralized bone product which exhibitsinferior osteoinductivity caused by excessive exposure of bone-inducingproteins in the bone to harsh acids over extended periods of time.

SUMMARY OF THE INVENTION

The present invention is directed to an apparatus for producingdemineralized osteoinductive bone. The apparatus achievesdemineralization of bone by subjecting bone to either a rapid highvolume, pulsatile acidification wave process or to a rapid continuousexchange of acid.

In one embodiment of the invention, the apparatus for demineralizingosteoinductive bone comprises a container for holding demineralizationsolution and the osteoinductive bone; a vessel cap covering thecontainer, the vessel cap containing a first port, and a second port forintroducing the osteoinductive bone into the container; a filter tubeassembly disposed within the first port for transporting thedemineralization solution into and out of the container, the filter tubeassembly being configured to exclude particles larger than a prescribedsize; a pump for removing the demineralization solution from thecontainer; and a first tube connecting the first port to the pump. Thefilter tube assembly preferably contains a plurality of openings along apredetermined portion thereof. Further, the filter tube assemblypreferably is configured to exclude particles larger than 300μ, morepreferably is configured to exclude particles larger than 225μ, and mostpreferably is configured to exclude particles larger than 125μ.

In accordance with the invention, a port filter assembly may be disposedwithin the second port for maintaining a sterile environment in theapparatus. The port filter assembly preferably provides a gas permeableseal. Further, the port filter assembly may comprise a fritted filterdisposed within an O-ring, the O-ring surrounded by a retaining ring.

The invention further provides that the container, the vessel cap, thefilter tube assembly, and the first tube are constructed from a materialhaving prescribed properties for preventing a chemical reaction with thedemineralization solution. For example, such material may be selectedfrom the group consisting of Teflon, glass, and ceramic.

In another embodiment of the invention, the apparatus for demineralizingosteoinductive bone further comprises a second tube connecting thesecond port to a vessel coupled to the pump. An ion exchange media isdisposed within the vessel for regenerating the demineralizationsolution removed from the container. The pump is preferably operated ata rate of about 0.25 to 4.0 liters per min., more preferably operated ata rate of about 0.5 to 2.0 liters per min., and most preferably operatedat a rate of about 1.0 liter per min. The ion exchange media maycomprise a strong cation exchange resin, a strong anion exchange resin,or a strong cation exchange resin and a strong anion exchange resin.

BRIEF DESCRIPTION OF THE FIGURES

The invention is further explained in the description which follows withreference to the figures and drawings, by way of non-limiting examples,various embodiments of the invention, with like reference legendsrepresenting similarly collected data throughout the several figures anddrawings.

FIG. 1. FIG. 1 illustrates a first embodiment of the inventive apparatusfor demineralizing bone according to the inventive process.

FIG. 2. FIG. 2 illustrates a side view of the inlet port filterassembly.

FIG. 3. FIG. 3 illustrates a second embodiment of the inventiveapparatus for demineralizing bone according to the inventive process.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions: The below definitions serve to provide a clear andconsistent understanding of the specification and claims, including thescope to be given such terms.

Acid. By the term “acid” is intended any acid or solutions containingone or more acids, capable of demineralizing bone. For example, suitableacids include highly ionizable acids including, but not limited to,hydrochloric acid, and weakly ionizable acids including, but not limitedto, citric acid. Suitable acids include, but are not limited to, organicacids such as formic acid, acetic acid, citric acid, or propionic acid;inorganic acids such as hydrochloric acid or phosphoric acid;physiological tissue-compatible hydroxy carboxylic acids including, butnot limited to, citric acid, gluconic acid, malic acid, tartaric acid,fumaric acid, and phosphoric acid; combinations of acids which chelate(bind) calcium and/or amino carboxylic agents including chelators whichchelate calcium including, for example, ethylenediaminetetraacetic acid(EDTA) (or analogues thereof), nitriloacetic acid (NTA), citric acid,succinic acid, and heparin. It has been found that these calciumchelating agent compositions, which bind calcium, aid in thedemineralization of bone by both organic and inorganic acids. Such acidsolutions may also include solutions of one or more acids in one or morealcohols. Any alcohol suitable for demineralizing bone such as, forexample, ethanol and isopropyl alcohol (IPA). Other suitable acidsolutions may include solutions of one or more acids in glycerol orother organic and/or inorganic metal remover, such as a metal chelator.Hydroxy carboxylic acids alone or in combination with amino carboxylicagents are advantageous for use in the demineralization process becausethey reduce the hydrolytic attack on bone morphogenic proteins presentin the bone and because they are antioxidants, which serve aspreservatives of the bone, thus eliminating the need for freeze dryingthe bone to preserve it.

Allowash™ Solution. By the term “Allowash™ solution” is intended for thepurposes of this invention to include, for example, those detergentcompositions disclosed in U.S. Pat. No. 5,977,034, incorporated hereinby reference. Examples of suitable Allowash™ compositions include acleaning composition containing about 0.06 wt % polyoxyethylene-4-laurylether; about 0.02 wt % poly (ethylene glycol)-p-nonyl-phenyl-ether;about 0.02 wt % octylphenol-ethyleneoxide and endotoxin freedeionized/distilled water.

Bone. By the term “bone” is intended, for the purposes of the invention,any bone as one having ordinary skill in the art would envision. Forexample, the bone includes autograft bone, allograft bone and xenograftbone. Moreover, such bone includes any bone from any source, including,for example, human bone and animal bone. The bone may be from a livingdonor or a cadaveric donor. The bone may include cortical bone and/orcancellous bone and/or cortical cancellous bone and may be present inany form including, for example, ground bone, particulate bone (i.e.dental bone), bone chips, bone strips, bone cubes, bone fibers, andessentially intact bone. The bone may be in any size. For example, theparticulate bone can be in any particle size range, such as, forexample, from about 120μ to about 860μ.

Bone Marrow Elements. By the term “bone marrow elements” is intended thehighly cellular hematopoietic connective tissue filling the medullarycavities and spongy epiphyses of bones which may harbor bacterial and/orviral particles and/or fungal particles, and includes, for example,blood and lipid.

Cycle. By the term “cycle” is intended one complete rotation of the trayof an orbital shaker, including, for example, orbital shaker byTroemner, Inc., Model 980001, Serial No: 1035, 500 watts. This orbitalshaker is preferably operated at a setting of from about 150 to about210 cycles/min., more preferably from about 160 to about 170cycles/min., which settings correlate to about from 20 to about 60cycles/min.

Detergent. By the term “detergent” is intended any agent which actsthrough a surface action, which possesses both hydrophilic andhydrophobic properties and/or exerts oil-dissolving (cleansing) and/orantibacterial and/or antiviral effects. Suitable detergents include, butare not limited to, anionic detergents, cationic detergents, acridinederivatives, long-chain aliphatic bases or acids, and Allowash™detergent solutions.

Disinfectant. By the term “disinfectant” is intended one or moredecontaminating agents which remove or inactivate/destroy any infectiousmaterial potentially present in the bone marrow of a bone graft. Forexample, potentially infectious material may include bacteria, virusand/or fungi. The disinfectant may include decontaminating agents suchas, for example, an antibacterial agent; an antiviral agent; anantimycotic agent; an alcohol including, for example, methyl, ethyl,propyl, isopropyl, butyl, an/or t-butyl; trisodium phosphate; sodiumhydroxide; hydrogen peroxide; and/or a detergent.

Drain. By the term “drain” is intended for the purposes of thisinvention, rapidly and substantially completely, draining or drawing offa volume of one or more demineralizing acids from a substantially closedprocessing container. Preferably, the demineralizing acid issubstantially completely drained from the processing container in lessthan 10.0 minutes, more preferably in less than 9.0 minutes.

Lipid. By the term “lipid” is intended the fat-soluble constituents ofbone marrow, including, for example, fatty acids, glycerides, andphospholipids.

Pulse. By the term “pulse” is intended for the purposes of thisinvention, rapidly and substantially completely, filling a substantiallyclosed processing container with a predetermined volume of one or moredemineralizing acids or acid solutions. Preferably, the container issubstantially completely filled with the predetermined volume ofdemineralizing acid in less than 3.0 minutes, more preferably in lessthan 2.0 minutes.

Solvent. By the term “solvent” is intended for the purposes of theinvention, a liquid cleaning composition capable of facilitating thesolubilization of lipid, facilitating bone marrow removal, inactivatingviral and/or bacterial particles, and/or disrupting cell membranes,and/or demineralizing bone. The liquid cleaning composition may containone or more of the following: water; saline; a detergent; adisinfectant; an acid; an alcohol, for example, ethanol and/orisopropanol; solvents; a combination of solutes desired to facilitatesolubilization of bone marrow, including, for example, Allowash™detergent solutions; a chelating agent; a bactericidal agent; anantimycotic agent; sodium hydroxide or similar strong base; organicand/or inorganic acid known and used in the art for the demineralizationof bone including, for example, hydrochloric acid; and/or hydrogenperoxide. Known lipophilic solvents include, for example, ethanol andchloroform.

Substantially Closed Processing Container. By the term “substantiallyclosed processing container” is intended for the purposes of the presentinvention, any rigid or deformable container or reservoir of a sizesufficient to contain bone and a predetermined volume of one or moredemineralizing acids, composed of a material that is stable when incontact with the demineralizing acids, and is configured to allow thecontinuous exchange or pulsed exchange of acid.

Undesirable Constituents. By the term “undesirable constituents” isintended for the purposes of the present invention any constituentsnormally associated with a particular tissue whose presence in thattissue to be transplanted is undesirable. Non-limiting examples include,for example, blood cells, bacteria, fungi, and viruses. In the case ofbone, bone marrow elements including lipid and blood, and any otherconstituents normally associated with bone marrow as well as anybacterial, viral or fungal contamination associated with the bone and/orbone marrow elements.

Ion Exchange Media. By the term “ion exchange media” is intended anymedia capable of removing calcium and/or from a demineralizing acidsolution, including, for example, 8% cross-linked DOWER 50WX8 50-100mesh, which is a cation exchange resin. Anion and cation resins areavailable with mesh sizes including 50-100, 100-200, and 200-400. Thereare three resin types, strong acid cation exchange resins designated as50W, Type I strong base anion exchange resins designated as 1, and TypeII strong base anion resins designated as 2. DOWER resins are fine meshresins (Dow Chemical Co., Midlant, Mich.) and microporous copolymers ofstyrene and divinylbenzene (DVB). Cross-linkage is measured by percentDVB content, and includes 2, 4, and 8. As one skilled in the art wouldappreciate, this enables selection of optimum levels of permeability,water retention capacity, and total capacity. Suitable ion exchangemedia include mixtures of cation and anion resins and include, forexample, one-third DOWER 50WX8 50-100 mesh, one-third DOWER 1 50-100mesh, and one-third DOWER 2 50-100 mesh; one-half DOWER 1 50-100 mesh,C1 form and one-half DOWER 50WX8 50-100 mesh, H form; and 200-400 meshat 1 liter per minute. However, it is appreciated that one skilled inthe art would be capable of selecting a suitable ion exchange resin toprocess bone.

Filter Mesh. By the term “filter mesh” is intended for the purposes ofthe invention any mesh composed of a material stable in the presence ofthe demineralizing solution having a mesh size sufficiently small so asto exclude bone particles. One skilled in the art is capable ofselecting a suitable filter mesh in accordance with the presentinvention. Suitable filter mesh includes polyester monofilament having amesh size of from 100μ to about 300μ, preferably from about 1 00μ toabout 225μ, and most preferably about 125μ. Such filter mesh includesPes125, (Industrial Fabrics Corp., Minneapolis, Minn.), which is apolyester monofilament mesh having a mesh size of 125μ.

The inventive process allows for the decalcification of an entire singledonor's tissue volume in a single vessel over a tissue weight range of100 to 800 grams or more at a rapid demineralization rate over a shortperiod of time. The bone produced according to this invention isuniformly demineralized and optimally osteoinductive.

II. Procurement and Processing of Bone

Bone is procured and processed according to methods well known in theart to which the invention pertains. For example, bone is procured froma cadaver donor, cleaned of soft tissue, and bone marrow elements andundesirable constituents are removed. The bone is then processed to adesired form including, for example, ground into particulate bone, cutinto cubes or strips, or left essentially intact. Bone is procured andprocessed under conditions according to accepted industry standards.Both cortical and/or cancellous bone is suitable for use in theinventive process.

III. Demineralization of Bone

Using the inventive process, bone is demineralized with acid at aconcentration sufficient to demineralize bone. For example, relativelystrong acids such as hydrochloric acid may be used at a concentration offrom about 0.1N to about 3.0N. Relatively weak acids including, forexample, citric acid, may be used at a concentration of from about 0.5Nto about 5.0N. The acid, for example, citric acid, may be dissolved inone or more lipid soluble alcohols containing permeation enhancementsurfactants to enhance the chemical reactivity and physical penetrationof the acid into the mineral apatite of the bone. Weak acids includingcitric acid may be used in combination with low concentrations of strongacids including, for example, hydrochloric acid, to provide ademineralization system in which a desired pH, for example, a pH ofabout 1.2 has been found to correlate to and not exceed a residualcalcium level of about 2.0 wt %, thus eliminating the potential ofover-decalcifying the bone matrix.

The rate of demineralization, i.e., grams of bone demineralized perminute, can be increased or decreased as desired, by one of ordinaryskill in the art to which the present invention pertains and withoutundue experimentation. As will be appreciated by an ordinary artisan,the rate of demineralization may vary based on factors which include:the reaction temperature; the concentration or normality of the acid andthe acid's neutralization potential (strong or weak) in reacting withCa⁺² hydroxy apatite; the acid's dissociation or percent ionization; thedelivery rate of the acid to the bone or the bone to the acid; the mass,volume and density of the bone to be demineralized; the concentration ofthe calcium hydroxyapatite in the bone; the degree to which the bone hasbeen cleaned of fat and protein; the surface area of the bone particlesand their particle size distribution; the compaction of the bone uponcontact with the acid by the action of the acid on the bone and the rateat which the products (calcium and phosphate) are removed from the acid;the method of agitation, i.e., mechanical stirring, shaking, orbitalshaking, sonication, as well as other methods of agitation which provideuniform concentration of the reacting species and reduction of boundarylayer resistance; and the degree to which a boundary layer resistanceforms on the microporous surface of the bone particle and the packing ofthese particles with each other. Accordingly, the demineralization ratecan be increased, for example, by increasing any one or more of theforegoing factors, for example, by increasing the temperature, acidconcentration, surface area of the bone to be demineralized, oragitation. Likewise, the demineralization rate can be decreased bydecreasing any one or more of the foregoing factors, for example,decreasing the acid concentration, slowing the delivery of acid, and/orincreasing bone particle size.

In accordance with the present invention, the demineralizing acidsolution may be pulsed into the ion exchange column containing the boneor continually pumped through an ion exchange column which removes boththe cations (calcium) and anions (phosphate). In the event of the pulsedexchange, calcium is removed and the acid is drained. In the event ofthe continuous exchange, calcium is removed and the acid is continuouslyregenerated. Suitable ion exchange media includes an 8% cross-linkedDOWER 50WX8 50-100 mesh, which will remove any calcium ions from diluteacid. The acid in the demineralizing acid solution competes with thecalcium for the binding sites. While any concentration suitable todemineralize bone may be used, it has been found that the lower theconcentration of acid, the greater the efficacy of calcium removal.Phosphate anion removal requires an anion exchange resin including, forexample, DOWER 1 and DOWER 2. These ion exchange media will maintain thepH of the bone-acid reaction mixture thus eliminating the necessity ofstopping the demineralization process in order to remove the solubilizedcalcium from the bone tissue. The ion exchange columns can beinactivated, for example, by a flow valve when it is time to wash thedemineralized bone at the end of the demineralization process. The ionexchange columns can be reused, re-sterilized and through the use of“selectivity charts” can be optimized for efficacy.

The rapid demineralization inventive process is stopped when a desiredresidual calcium level of calcium in the bone matrix being demineralizedhas been reached. U.S. Pat. Nos. 6,189,537 and 6,305,379 are directed tomethods for producing osteoinductive bone and the osteoinductive boneproduced thereby, and are hereby incorporated herein by reference intheir entirety. To determine a stopping point, a particular pH of eluentacid (exiting the reaction chamber prior to being run through an ionexchange media during recirculation), which correlates with the desiredresidual calcium level, must be determined. This is done by firstobtaining a bone sample and determining the initial calciumconcentration of the bone according to methods well known in the art towhich the invention applies; demineralizing bone at a constant rate;simultaneous with demineralizing, periodically sampling the eluent acidsolution and the bone from the closed reaction container at specificintervals of time during the demineralization process; determining thepH of each sample of acid solution and determining the residual calciumlevel of each corresponding bone sample; plotting the pH of a sampleversus the calcium concentration of the corresponding bone sample, anddrawing a curve; and from the curve determining what pH of the acidcorrelates with the desired residual calcium level. Thereafter, theresidual calcium level of a bone sample can be determined by determiningthe pH of a sample of the acid solution, sampled at a time point duringdemineralization of the bone sample, by determining the calciumconcentration on the curve which corresponds to the pH of the acidsample.

The amount of acid needed is that sufficient to demineralize bone to adesired residual level and may be determined according to known methods.In a preferred embodiment, the weight of the ground bone is firstdetermined, the donor weight in grams is divided by 100 grams, and theresultant number is either multiplied by 3 liters for pulsed aciddemineralization or multiplied by one liter for continuous aciddemineralization. This is the total volume of acid needed todemineralize the given amount of ground bone. Other acids and desiredcalcium levels can be used by monitoring the calcium levels duringdemineralization at specific time points and plotting a curve todetermine how much acid is used to reach a specific calcium level.

In the event of pulsed acid demineralization, the number of pulse/draincycles needed may be calculated according to known methods. The totalvolume of acid needed to demineralize the given amount of bone iscalculated and then divided by 4. This number is the number ofpulse/drain cycles needed. Each pulse/drain cycle is carried out with 4liters of acid.

For example, if the amount of ground bone is 425 grams, this number isdivided by 100 grams to equal 4.25, which is then multiplied by 3 litersto yield a total acid volume of 12.75 liters. This number is thendivided by 4 to yield 3.19 pulse/drain cycles. Thus, for 425 grams ofbone, 12.75 liters of acid is needed, and processing includes 3pulse/drain cycles using 4 liters each, and a fourth pulse/drain cycleusing 0.75 liters of acid (the remainder).

In this calculation, there must be at least two pulse/drain cycles, andeach cycle must include at least 5.0 min. incubation. This calculationis specific for 0.5N HCl and for reaching 2% residual calcium.

IV. Apparatus for Demineralizing Bone

FIG. 1 illustrates the apparatus 1 of the invention for pulsed aciddemineralization of bone. Bone is demineralized by placing bone, forexample, ground bone, in container 3 holding a demineralizationsolution, for example, one or more acids at a concentration sufficientto demineralize bone, and in a volume sufficient to process the amountof bone to be demineralized, for example, from about 2.0 to about 8.0liters, preferably from about 3.0 to about 6.0 liters of acid. Thecontainer 3 optionally includes heating blanket or thermal wrap 2.

Thereafter, a defoaming agent is added to the container through tubing 4connected to a first port 6 on the vessel cap 5. Vessel cap 5 isconnected to container 3 via O-rings 13. The defoamer flows intocontainer 3 through filter tube assembly 7 disposed within the firstport 6. Filter tube assembly 7 includes the filter mesh 8 disposed overthe filter tube 9 optionally having openings 10, and a downtube 21optionally having openings 21 a (at least two). The filter tube assembly7 is connected at its top end to a vessel cap 5 via a connection 11 andtop end cap 11 a. This connection can be a threaded connection, africtional connection, a connection via O-rings, or the equivalent. Thefilter tube assembly 7 is connected at its bottom end to end cap 14 via,for example, a press fit, a frictional fit, a threaded connection, orvia O-rings or the equivalent. The filter tube assembly 7 at its bottomend may be seated on protrusion 12 of container 3. The keeper rings 15may be used to anchor filter mesh 8 to filter tube 9.

The filter mesh 8 may be composed of any material stable in the presenceof the demineralizing solution, such as polyester or Teflon, or anequivalent material. The filter mesh may be any size suitable to excludebone particles. In a preferred embodiment, the mesh size is about 125μ.

Suitable defoamers include any defoamers well known in the art to whichthe invention pertains, and include, for example, ethanol. Any amount ofethanol suitable for defoaming, such as 60 mls of 200 proof ethanol in6.0 liters of acid, may be used. The bone-acid-ethanol solution is thenpreferably vigorously agitated by known methods. For example, the closedapparatus may be exposed to orbital shaking at a rate sufficient to keepthe bone particles in suspension, for example, at from about 20 to about60 cycles per minute on an orbital shaker table, while the system ismaintained at a desired temperature, for example, of from about 15° C.to about 100° C., preferably from about 15° C. to about 50° C., morepreferably from about 20° C. to about 40° C., and most preferably atabout 23° C.

After an initial period of time, for example, five minutes,substantially all, for example 90% to 95%, of the acid is then rapidlydrained from container 3 through tubing 4 connected to the first port 6on vessel cap 5, via a vacuum pump connected to tubing 4. The acidsolution is thus rapidly pulled through the first port 6, through filtermesh 8 covering filter tube 9 of filter assembly 7, and downtube 21having openings 21 a, and exiting the container 3 via tubing 4.Container 3 was then refilled with the demineralizing solution, viatubing 4 connected to the first port 6 on vessel cap 5, and flowing intocontainer 3 through downtube 21 and filter mesh 8 covering filter tube 9of filter assembly 7. The contents of the container 3 were againagitated for a second period of time, for example, about ten minutes.The container 3 was optionally again rapidly drained, refilled, andagitated for a third period of time, for example, 20 minutes.

The acid was then drained and a buffer solution was added to container 3through tubing 4 connected to vessel cap 5 and filter assembly 7, tostop the demineralizing reaction. Vessel cap 5 further includes a secondport 16 used to fill the reaction vessel 2 with bone. Thereafter, portfilter assembly 17, illustrated in FIG. 2, including retaining ring 18,O-ring 19, and fritted filter 20, is placed in the port to allowmaintenance of the closed system during pulse and drain exchanges. Gascan sterilely leave or enter container 3 via port filter assembly 17.All of the components of apparatus 1 can be composed of any materialstable in the presence of the demineralizing solution. Suitablematerials include Teflon, glass, and ceramic.

FIG. 3 illustrates the apparatus 22 of the invention for continuous aciddemineralization of bone. Bone is demineralized by placing bone, forexample, ground bone, in reaction vessel 23 containing ademineralization solution, for example, one or more acids at aconcentration sufficient to demineralize bone, and in a volumesufficient to process the amount of bone to be demineralized, forexample, from about 2.0 to about 10.0 liters, preferably from about 3.0to about 7.0 liters of acid.

Thereafter, a defoaming agent is added to the vessel 23 through inflowtubing 27 connected to inlet port 26. Suitable defoamers include anydefoamers well known in the art to which the invention pertains, andinclude, for example, ethanol. Any amount of ethanol suitable fordefoaming, such as 60 mls of 200 proof ethanol in 6.0 liters of acid,may be used. The bone-acid-ethanol solution is then preferablyvigorously agitated by known methods. The closed apparatus may bestirred at from about 500 rpm to about 2500 rpm, preferably 1000 rpm toabout 2000 rpm, and more preferably stirred at about 1350 rpm withmixing paddle 28, for example Cole Palmer Model No: E-04541-00 303/304supplied by Cole Palmer Instrument Co., Vernon Hills, Ill. The closedapparatus 22 may also be agitated by orbital shaking while the system ismaintained at a desired temperature, for example, of from about 0° C. toabout 100° C., preferably from about 15° C. to about 50° C., morepreferably from about 20° C. to about 40° C., and most preferably atabout 23° C.

The demineralizing acid solution is continuously exchanged by pumpingthe acid solution from the reaction vessel 23, through outlet port 24,through outflow tubing 25, which tubing runs through pump 28. Pump 28 isoperated at from 0.25 to 4.0 liters per min., preferably 0.5 to 2.0liters per min, and most preferably about 1.0 liter per min. The eluentacid solution is delivered to ion exchange media vessel 29 through inlet30. As the acid solution is continually pumped and calcium and phosphateare removed from the acid solution by the ion exchange media 32, theregenerated acid exits the ion exchange media at outlet 31 and flowsback into the reaction vessel 23 through inflow tubing 27. The ionexchange media vessel 29 is disposed on a magnetic stir plate to stirthe ion exchange media 32 during the process. The reaction vessel 23 isdisposed on an orbital shaker to agitate the acid/bone mixture duringprocessing.

All of the publications cited herein are hereby incorporated byreference into the present disclosure. It will be appreciated by thoseskilled in the art to which the invention pertains that variousmodifications can be made without departing from the essential naturethereof. It is intended to encompass all such modification within thescope of the appended claims.

1. An apparatus for demineralizing osteoinductive bone, comprising: acontainer for holding demineralization solution and the osteoinductivebone; a vessel cap covering said container, said vessel cap containing afirst port, and a second port for introducing the osteoinductive boneinto said container; a filter tube assembly disposed within said firstport for transporting said demineralization solution into and out ofsaid container, said filter tube assembly being configured to excludeparticles larger than a prescribed size; a pump for removing saiddemineralization solution from said container; and a first tubeconnecting said first port to said pump.
 2. The apparatus of claim 1,wherein said filter tube assembly contains a plurality of openings alonga predetermined portion thereof.
 3. The apparatus of claim 1, furthercomprising a port filter assembly disposed within said second port formaintaining a sterile environment in said apparatus.
 4. The apparatus ofclaim 3, wherein said port filter assembly provides a gas permeableseal.
 5. The apparatus of claim 3, wherein said port filter assemblycomprises a fritted filter disposed within an O-ring, said O-ringsurrounded by a retaining ring.
 6. The apparatus of claim 1, whereinsaid filter tube assembly is configured to exclude particles larger than300μ.
 7. The apparatus of claim 1, wherein said filter tube assembly isconfigured to exclude particles larger than 225μ.
 8. The apparatus ofclaim 1, wherein said filter tube assembly is configured to excludeparticles larger than 125μ.
 9. The apparatus of claim 1, wherein saidcontainer, said vessel cap, said filter tube assembly, and said firsttube are constructed from a material having prescribed properties forpreventing a chemical reaction with said demineralization solution. 10.The apparatus of claim 9, wherein said material is selected from thegroup consisting of Teflon, glass, and ceramic.
 11. The apparatus ofclaim 1, further comprising a second tube connecting said second port toa vessel coupled to said pump.
 12. The apparatus of claim 11, wherein anion exchange media is disposed within said vessel for regenerating saiddemineralization solution removed from said container.
 13. The apparatusof claim 11, wherein said pump is operated at a rate of about 0.25 to4.0 liters per min.
 14. The apparatus of claim 11, wherein said pump isoperated at a rate of about 0.5 to 2.0 liters per min.
 15. The apparatusof claim 11, wherein said pump is operated at a rate of about 1.0 literper min.
 16. The apparatus of claim 12, wherein said ion exchange mediacomprises a strong cation exchange resin.
 17. The apparatus of claim 12,wherein said ion exchange media comprises a strong anion exchange resin.18. The apparatus of claim 12, wherein said ion exchange media comprisesa strong cation exchange resin and a strong anion exchange resin.
 19. Anapparatus for demineralizing osteoinductive bone, comprising: acontainer for holding demineralization solution and the osteoinductivebone; a vessel cap covering said container, said vessel cap containing afirst port, and a second port for introducing the osteoinductive boneinto said container; a filter tube assembly disposed within said firstport for transporting said demineralization solution into and out ofsaid container, said filter tube assembly being configured to excludeparticles larger than a prescribed size, and said filter containing aplurality of openings along a predetermined portion thereof; a pump forremoving said demineralization solution from said container; and a firsttube connecting said first port to said pump.
 20. An apparatus fordemineralizing osteoinductive bone, comprising: a container for holdingdemineralization solution and the osteoinductive bone; a vessel capcovering said container, said vessel cap containing a first port, and asecond port for introducing the osteoinductive bone into said container;a filter tube assembly disposed within said first port for transportingsaid demineralization solution into and out of said container, saidfilter tube assembly being configured to exclude particles larger than125μ, and said filter containing a plurality of openings along apredetermined portion thereof; a pump for removing said demineralizationsolution from said container; and a first tube connecting said firstport to said pump.
 21. An apparatus for demineralizing osteoinductivebone, comprising: a container for holding demineralization solution andthe osteoinductive bone; a vessel cap covering said container, saidvessel cap containing a first port, and a second port for introducingthe osteoinductive bone into said container; a filter tube assemblydisposed within said first port for transporting said demineralizationsolution into and out of said container, said filter tube assembly beingconfigured to exclude particles larger than a prescribed size; a pumpfor removing said demineralization solution from said container; a firsttube connecting said first port to said pump; and a second tubeconnecting said second port to a vessel coupled to said pump, wherein anion exchange media is disposed within said vessel for regenerating saiddemineralization solution removed from said container.
 22. An apparatusfor demineralizing osteoinductive bone, comprising: a container forholding demineralization solution and the osteoinductive bone; a vesselcap covering said container, said vessel cap containing a first port,and a second port for introducing the osteoinductive bone into saidcontainer; a filter tube assembly disposed within said first port fortransporting said demineralization solution into and out of saidcontainer, said filter tube assembly being configured to excludeparticles larger than 125μ, and said filter containing a plurality ofopenings along a predetermined portion thereof; a pump for removing saiddemineralization solution from said container; a first tube connectingsaid first port to said pump; and a second tube connecting said secondport to a vessel coupled to said pump, wherein an ion exchange media isdisposed within said vessel for regenerating said demineralizationsolution removed from said container.